Tobacco nitrosamine NNK increases ALDH-positive cells via ROS-Wnt signaling pathway in A549 human lung cancer cells

Abstract

-Epidemiological studies suggest that lung cancer, which is a major cause of cancer death, has a critical association with cigarette smoking. Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in cigarette smoke is a major risk factor for carcinogenesis. However, the mechanisms by which NNK promotes cancer development have not been fully elucidated. Growing evidence suggests that lung cancer originates from cancer stem cells (CSCs), which are a minor population of lung cancer cells. In the present study, we investigated the effects of NNK on the CSCs in A549 human lung cancer cells using flow cytometry with aldehyde dehydrogenase (ALDH), a functional marker of CSCs. We found that NNK increased the proportion of ALDH-positive cells in a dose-dependent manner. A Wnt inhibitor PNU74654 reduced NNK-induced expression levels of Wnt target gene Dkk1 and increase in ALDH-positive cells. We next examined the signaling pathway that mediates the NNKinduced increase in ALDH-positive cells via Wnt signaling. DCF assay revealed that NNK induced reactive oxygen species (ROS) production. The ROS scavenger N-acetylcysteine (NAC) inhibited the NNKinduced Wnt activation and increase in ALDH-positive cells. These data suggest that NNK-induced ROS activate the Wnt signaling pathway in A549 cells. These findings would provide new insights into the role of NNK in the lung CSCs.

The Effect of NNK, A Tobacco Smoke Carcinogen, on the miRNA and Mismatch DNA Repair Expression Profiles in Lung and Head and Neck Squamous Cancer Cells

“…Cancer cells reached 70-80% confluency and were then exposed to experimental media for 24 h. Experimental groups included exposure to (i) 1 µM and (ii) 2 µM of 4-(N-Methyl-N-Nitrosamino)-1-(3-pyridyl)-1-butanone (CAS 64091- or NNK (sc-209854 Santa-Cruz ® , Dallas, TX, USA) [17] (NNK dissolved in DMSO-vehicle as 1 M stock solution). Cells were incubated in serum-free medium (EMEM for FaDu and RPMI-1640 for NCI, with 1% pen/strep) with NNK, at 37 • C in humidified air and 5% CO 2 .…”

“…It is believed that exposure of cells to tobacco smoke carcinogens can lead to DNA damage, which may cause chromosomal instability and increased cell proliferation [10][11][12][13]. In particular, tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which is one of the chemicals in tobacco smoke, has been linked to lung and head and neck cancer [14], and has also been shown to upregulate oncogenic pathways [15][16][17].The development and progression of lung and head and neck malignancies appear to be a complex process. Although multiple diagnostic and prognostic markers have been identified for both lung and head and neck cancers [18,19], the precise molecular mechanisms involved in the development and progression of these malignancies remain unclear.We understand that a functional DNA repair mechanism that includes the recognition and repair of mismatch DNA errors during DNA replication is essential in eliminating the harmful effect of several environmental risk factors, such as NNK, on the exposed cells [20][21][22][23].…”

“…It is believed that exposure of cells to tobacco smoke carcinogens can lead to DNA damage, which may cause chromosomal instability and increased cell proliferation [10][11][12][13]. In particular, tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which is one of the chemicals in tobacco smoke, has been linked to lung and head and neck cancer [14], and has also been shown to upregulate oncogenic pathways [15][16][17].…”

Whole exome sequencing approach to analysis of the origin of cancer stem cells in patients with head and neck squamous cell carcinoma

“…The number of global variants identified in the tumour cells was higher than in the CSC ALDH + (1402), indicating high heterogeneity by these cells groups. The heterogeneity of CSCs may be being brought about by intrinsic differences such as genetic mutations, epigenetic changes, cell–cell interactions and microenvironmental differences as chemotactic factors, and hypoxia conditions …”

“…Remarkably, that patient was the only smoker in the study. Several studies support that cigarette smoke might severely affect mobilization, trafficking and homing of stem cells, and even can increases ALDH‐positive cells via the ROS‐Wnt signalling pathway in A549 human lung cancer cells …”